Fluidic temperature gradient focusing

ABSTRACT

A method and device are provided for concentrating and separating ionic species in solution within a fluidic device having a fluid conduit such as a channel or capillary. The concentration is achieved by balancing the electrophoretic velocity of an analyte against the bulk flow of solution in the presence of a temperature gradient. Using an appropriate buffer, the temperature gradient can generate a corresponding gradient in the electrophoretic velocity so that the electrophoretic and bulk velocities sum to zero at a unique point and the analyte will be focused at that point. The method and device may be adapted for use with a variety of analytes including fluorescent dyes, amino acids, proteins, DNA and to concentrate a dilute analyte.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of the filing date of bothcopending Provisional Patent Application No. 60/307,691, filed on Jul.25, 2001, and 60/323,404, filed on Sep. 19, 2001.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This invention was made by employees of the United StatesGovernment and may be manufactured and used by or for the Government forgovernmental purposes without the payment of any royalties.

FIELD OF THE INVENTION

[0003] The present invention relates to a method for electrokineticfocusing of samples, and in particular, methods for electro-focusingsamples in fluidic devices using electric field gradients.

BACKGROUND OF THE INVENTION

[0004] Over the past decade a great deal of research has been focused onthe development of technology related to micro-total-analytical systems.This technology is based on the concept of a series of microfluidicchannels also known as microchannels for the movement, separation,reaction, and/or detection of various chemicals or biological compoundssuch as amino acids, proteins, and DNA.

[0005] One disadvantage with prior microfluidic devices is that there isfrequently a mismatch between the extremely small quantities of sampleused for analysis and the often much larger quantities needed forloading the sample into the microfluidic device and transporting thesample to the point of analysis. For example, a typical analysis samplemay be around one nanoliter or less of a liquid containing sample thatis injected into a separation channel and then separatedelectrokinetically as it moves down the channel to a detection region.However, the channels used to transport the sample to the injectionpoint are typically also filled with the sample, thus increasing therequired amount of the sample by a factor of 100 or more. In addition,the sample is typically loaded onto the microfluidic device into areservoir from a pipette so that in all, approximately 99.9% of thesample is discarded as waste.

[0006] Electric field gradient focusing is one way of addressing theproblem of requiring a large sample for analysis due to theinefficiencies of conventional devices which result in wasted sample.Electric field gradient focusing can be used to concentrate samples at agiven point within a microfluidic device before the analysis step.Further, the electric field gradient can be used to concentrate all ofthe sample at the beginning of the separation channel so that verylittle of the sample would be wasted.

[0007] Electric field gradient focusing is accomplished by theapplication of an electric field gradient within a microchannel. Inresponse to the electric field gradient, there is a correspondinggradient in the electrophoretic velocity of any ion within themicrochannel. The total velocity of the ion is the sum of itselectrophoretic velocity and the bulk fluid velocity. If these twocomponents of the velocity are in opposite directions, they can bebalanced so that the molecule will have zero total velocity.

[0008] When there is a gradient in the electrophoretic velocity, thebalance between bulk and electrokinetic velocities can occur at a singlepoint within the microchannel and therefore can result in focusing ofions at that point. Typically, the electric field gradient used infocusing is generated by the external manipulation of the electric fieldin the middle of the microchannel through the use of conducting wires,salt bridges, porous membranes, or other structures that will passelectric current but will restrict the flow of bulk fluid and analytesthat are to be focused.

[0009] Several recent developments with regard to focusing methods inmicrofluidics, and in particular, the use of electric field gradients,have been made. A description of related methods of focusing can befound in C. F. Ivory, W. S. Koegler, R. L. Greenlee, and V. Surdigio,Abstracts of Papers of the American Chemical Society 207, 177-BTEC(1994); C. F. Ivory, Separation Science and Technology 35, 1777 (2000);Z. Huang and C. F. Ivory, Analytical Chemistry 71, 1628 (1999); W. S.Koegler and C. F. Ivory, Journal of Chromatography a 726, 229 (1996);and P. H. Ofarrell, Science 227, 1586 (1985), all of which are herebyincorporated by reference.

[0010] To illustrate the basic principles disclosed in thesepublications, reference is made to FIG. 1(a) which depicts a length ofbuffer-filled microchannel of constant cross-sectional area with anelectrode, denoted 4, in the middle, and two further electrodes at eachend, denoted 3 and 5, so that the voltages V₁, V₃ at the ends and thevoltage V₂ at the middle of the channel can be controlled. A singlespecies of negatively charged analyte is present in a buffer that isprovided to the microchannel. The electrical connection, represented aselectrode 4, can be accomplished with a simple metal wire as depicted inFIG. 1(a), or through a more complicated structure consisting ofadditional fluid channels and porous membrane structures or saltbridges.

[0011] The electric field in the section 1, i.e., the channel betweenelectrodes 3 and 4 is E₁=(V₂−V₁)/(l/2) and the electric field in section2, i.e., between electrodes 4 and 5, is E₂=(V₃−V₂)/(l/2), where V₁, V₂,and V₃ are the voltages applied to the three electrodes 3, 4, and 5, andl is the length of the microchannel. If, E₁ differs from E₂ as shown inFIG. 1(b), the electrophoretic velocity of the analyte in the channel,u_(E1′), will be different in section 1 than in section 2. If an overallbulk fluid velocity, u_(B)<0, is applied, e.g., either electro-osmoticor pressure-driven, the bulk fluid velocity must be the same, due tocontinuity, in all parts of the microchannel. The total velocity of theanalyte, u_(T)=u_(B)+u_(EP), will then be the sum of the electrophoreticand bulk velocities, which can differ in section 1 from section 2.

[0012] The use of the microchannel device of FIG. 1(a) for focusing ofthe ions is illustrated in FIG. 2 where u_(T, 1)>0>u_(T, 2), so that theions flow into the middle from both directions and are thus focused inthe middle of the channel near electrode 4.

[0013] One major drawback to electric field gradient focusing is thatthe microchannel device tends to be difficult to construct and that itrequires the control of voltage on an additional electrode, e.g. 4 ofFIG. 1(a), that is used to apply the electric field gradient. Inaddition, if electrodes are used to generate electric field gradients,unwanted chemical products will be generated electrochemically at thebuffer-electrode interface. If the electric field gradient is producedthrough the use of a salt bridge or membrane, the electrochemicalproducts can be avoided, however only chemical species that cannot passthrough the membrane or salt bridge can be focused.

[0014] Two additional methods for concentrating a sample include samplestacking and field amplified sample injection in which a sample isconcentrated as the sample crosses a boundary between low and highconductivity buffers. These methods can achieve preconcentration factorsof 100 to 1000-fold although these methods require multiple buffers.Sweeping is yet another concentration method which is capable of a veryhigh degree of sample concentration (e.g., up to 5000-fold), but isuseful only for small hydrophoic analytes with a high affinity for amobile micellular phase.

[0015] An additional technique for concentrating an ionic sampleincludes isoelectric focusing. Isoelectric focusing is commonly used forthe concentration and separation of proteins and involves the focusingof analytes at their respective isoelectric points (pls) along a pHgradient.

[0016] Two examples of recent isoelectric focusing techniques areprovided by U.S. Pat. Nos. 3,664,939 to Luner et al. and 5,759,370 toPawliszyn. Both references relate to isoelectric focusing with pHgradients that are created by the application of a temperature gradient.The isoelectric focusing uses a pH gradient to focus analytes and inparticular proteins, at their isoelectric points. The isoelectric pointis the pH at which the analyte has zero electrophoretic mobility, i.e.,approximately zero charge. pH gradients for isoelectric focusing aretypically generated using ampholyte mixtures or immobilized ampholytesin gels. The two above referenced patents are included here as examplesof prior art uses of temperature gradients for focusing. It is actuallyvery unusual for isoelectric focusing to be done with a pH gradientgenerated with using a temperature gradient.

[0017] One disadvantage with isoelectric focusing is that it is limitedin application because it can only be used with analytes with anaccessible pl. Additionally, the concentration to which a protein can befocused with isoelectric focusing is severely limited due to the lowsolubility of most proteins at their pls.

BRIEF SUMMARY OF THE INVENTION

[0018] The present invention concerns a method and device forconcentrating and separating ionic species in solution within fluidconduits which include channels, microchannels, and capillary tubes. Theconcentration is achieved by balancing the electrophoretic velocity ofan analyte against the bulk flow of solution in the presence of atemperature gradient. Using an appropriate buffer, the temperaturegradient can generate a corresponding gradient in the electrophoreticvelocity so that the electrophoretic and bulk velocities sum to zero ata unique point and the analyte will be focused at that point. Thepresent invention may be adapted for use with any charged analyte,including fluorescent dyes, amino acids, proteins, DNA, cells, andparticles and may provide up to or, in some instances, exceed a10000-fold concentration of a dilute analyte.

[0019] One aspect of the present invention concerns a method fordirecting ionic analytes contained in an ionic buffer solution of asystem and which may include concentrating or separating analytespresent in the buffer solution. The method includes producing anelectric current flow in an ionic buffer solution containing at leastone species of ionic analyte to cause the analyte ions to migrateelectrophoretically. A temperature gradient is established in the buffersolution to have a significant component substantially aligned with thecurrent flow, to thereby generating a gradient of the electrophoreticvelocity of the analytes. A bulk flow is produced in the buffer solutionsuch that the bulk flow has a significant component substantiallyaligned in the direction opposite the direction of the electrophoreticmigration of one or more of the analytes so that the total velocity ofone or more of the analytes is equal to zero at some point in thesystem.

[0020] According to another aspect of the present invention, a fluidicdevice includes a fluid conduit and an ionic buffer disposed in theconduit. At least one source or sink of heat, thermally coupled to thefluid conduit, is provided for establishing a temperature gradienthaving a significant component substantially aligned with the currentflow so as to form an electrophoretic velocity gradient within the fluidconduit. A voltage potential source is provided for applying an electricfield along a length of the fluid conduit and a current source providesan electric current flow through the ionic buffer in the fluid conduit.A source of bulk fluid flow provides for an opposing flow of the bufferin the fluid conduit. In alternate, further embodiments, the ionicbuffer has either a temperature dependent ionic strength or atemperature dependent pH such that when a temperature gradient isapplied to the fluid conduit, an electrophoretic velocity gradient isestablished in the ionic buffer present in the fluid conduit.

[0021] One advantage or feature of the present invention is provided bya technique that allows for simultaneous concentration and separation ina manner similar to isoelectric focusing but which is adoptable for usewith any charged analyte and is not limited to molecules for a specificpl or range of pls. Further, the temperature gradient focusing of thepresent invention can be used to achieve higher degrees of sampleconcentration, e.g., more than 10,000 fold concentration of a dilutesample, when compared with any prior single sample preconcentrationmethod.

[0022] A further feature of the present invention is that theelectrophoretic velocity gradient is formed within the channel orcapillary in response to the temperature gradient without the need forexternally manipulated voltages or complicated and difficult tofabricate semi-permeable structures.

[0023] Further features and advantages of the present invention will beset forth in, or apparent from, the detailed description of preferredembodiments thereof which follows.

BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The invention will now be described in detail with respect topreferred embodiments with reference to the accompanying drawings,wherein:

[0025]FIG. 1(a) is a schematic depicting a prior art microchannel devicewhich provides for electric field gradient and FIG. 1(b) is a plot ofthe electric field versus distance (x) along the microchannel of FIG.1(a);

[0026]FIG. 2 is a plot of velocity versus distance along themicrochannel of FIG. 1(a);

[0027]FIG. 3(a) is a schematic illustration of temperature gradientfocusing and fluid conduit in the form of a microchannel in accordancewith the present invention, FIG. 3(b) depicts temperature distributionalong the microchannel of FIG. 3(a), and FIG. 3(c) is a plot of thefunction${f(T)} = \frac{{\sigma (20)} \cdot {\eta (20)}}{{\sigma (T)} \cdot {\eta (T)}}$

[0028] plotted as a function of the distance along the microchannel ofFIG. 3(a), and FIG. 3(d) is a plot depicting velocity as a function ofdistance along the microchannel;

[0029]FIG. 4(a) is a schematic illustration of a microchannel fortemperature gradient focusing created by Joule heating according toanother embodiment of the present invention, FIG. 4(b) depicts thetemperature profile along a length of the microchannel of FIG. 4(a),FIG. 4(c) depicts the electric field profile along a length of themicrochannel of FIG. 4(a), and FIG. 4(d) is a plot showingelectrophoretic velocity, bulk velocity, and total velocity vs. distancealong the microchannel of FIG. 4(a);

[0030]FIG. 5 is a schematic drawing of a fluidic device according tofurther embodiment of the present invention; and

[0031]FIG. 6 is a schematic drawing of a capillary fluidic deviceaccording to an alternate embodiment of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

[0032] The present invention provides temperature gradient focusing of asample in a fluidic device which includes a fluid conduit such as achannel or capillary tube. Temperature gradient focusing focusesanalytes by balancing the electrophoretic velocity of an analyte againstthe bulk velocity of the buffer containing the analyte. If there is anappropriate gradient in the electric field, the total velocity of agiven charged analyte, as determined by the sum of the bulk andelectrophoretic velocities, can be set to zero at any point along thechannel and all the analyte in the system is moved toward that point.However, in contrast to electric field gradient focusing where theelectric field gradient is applied using a combination of electrodes andmembranes, using temperature gradient focusing of the present invention,the necessary electric field gradient is produced by the application ofa temperature gradient.

[0033] Further description of the present invention will now be madewith reference to the drawings, and in particular to FIG. 3(a), where abuffer-filled microchannel 10 includes electrode connections 12, 14 ateach end. The velocity of an analyte in the microchannel 10 is given bythe sum of its electrophoretic velocity, u_(EP), and the bulk velocity,u_(B), of the buffer:

u _(T) =u _(EP) +u _(B).

[0034] If there is a gradient in the electrophoretic velocity, the bulkvelocity can be adjusted so that the total velocity is equal to zero ata single point along the channel, and the analyte will be focused atthat point. The electrophoretic velocity of an analyte in themicrochannel 10 is given by the product of the electric field, E, andthe electrophoretic mobility of the analyte: u_(EP)=E·μ_(EP).

[0035] A temperature gradient is applied along the length of the channelas shown in FIG. 3(b). This results in corresponding gradients in boththe electric field E and the electrophoretic mobility μ_(EP).

[0036] The electric field in the microchannel 10 is given by:${E = \frac{I}{A \cdot \sigma}},$

[0037] where I is the electric current running through the microchannel10, A is the channel cross-sectional area of the microchannel 10, and σis the conductivity of the buffer. Since the conductivity of the bufferis temperature-dependent, the electric field is alsotemperature-dependent. Here, constant current is presumed because thecurrent running through any given section of the microchannel 10 will bethe same for all parts of the microchannel, whereas the voltage dropacross a portion of the microchannel 10 and the electric field in themicrochannel 10 will depend on the temperature of that portion. Oneskilled in the art will readily appreciate that the present temperaturegradient focusing differs from electric field gradient focusing in thatin electric field gradient focusing, the velocity gradient that is usedfor focusing results from a gradient in the electric field imposed bythe addition or subtraction or current from point or points within themicrochannel.

[0038] Using microchannel 10, it is possible to manipulate theconductivity of the buffer by changing the temperature. Consequently, itis possible to produce electric field gradients in microfluidic devices,such as microchannel 10, through the application of a temperaturegradient.

[0039] At fixed current density, the electric field in microchannel 10is inversely proportional to the conductivity of the buffer solution inthe microchannel. Most often, the primary temperature dependence of theconductivity is due to the variation of the solvent viscosity withtemperature, so it can be written as σ=σ₀·η(20)/(η(T)·ƒ(T)), where σ isthe conductivity, σ₀ is a constant, η(T) is the temperature dependentviscosity, and ƒ(T) is a function that accounts for any othertemperature dependence. Similarly, the temperature dependence of theelectric field is given by E=E₀·η(T)·ƒ(T)/η(20), where E is the electricfield and E₀ is a constant.

[0040] For most buffers, the function ƒ(T) is constant or only weaklydependent on temperature. However, it can be non-constant, i.e.,variable, if, for example, the ionic strength of the buffer istemperature dependent. Advantageously, the buffers of the presentinvention are characterized by a non-constant ƒ(T).

[0041] The electrophoretic mobility of an ionic (e.g., analyte) speciesin the buffer is also dependent on the viscosity, and so can be writtenas μ_(EP)=μ₀·η(20)/(η(T)·ƒ_(EP)(T)), where μ₀ and ƒ_(EP)(T) are definedin analogy to σ₀ and ƒ(T) where, for most analytes, ƒ_(EP)(T) will beconstant. The electrophoretic velocity of the analyte can then bewritten as u_(EP)=E₀·μ₀·ƒ(T)/ƒ_(EP)(T). It should be noted that if ƒ(T)and ƒ_(EP)(T) have the same temperature dependence, e.g., they are bothconstant, then u_(EP) will not be temperature dependent, and an electricfield gradient produced in this way can not be used for focusing.

[0042] If, on the other hand, ƒ(T) and ƒ_(EP)(T) do not have the sametemperature dependence, then temperature gradients will result ingradients in the electrophoretic velocity, which can be used forfocusing as described above.

[0043] One skilled in the art will readily appreciate a major advantageof this present method over some other methods of preconcentration isthat the concentration of the buffer salts is completely unaffected bythe focusing. This results from the fact that if the buffer salt isconsidered as an analyte, then, by definition, ƒ_(EP)(T)=ƒ(T) and thereis no gradient in the electrophoretic velocities of the buffer salts.

[0044] Most commonly this technique would be implemented with a buffercharacterized by a strongly temperature dependent ƒ(T) and with analytescharacterized by a constant or nearly constant ƒ_(EP)(T). However, thepresent temperature gradient focusing can also be implemented in asystem in which ƒ(T) is constant and ƒ_(EP)(T) is not, or in which bothƒ(T) and ƒ_(EP)(T) are non-constant, but differ in their temperaturedependence.

[0045] The counterbalancing bulk flow can be applied electroosmoticallyif the electro-osmotic mobility does not differ too much from theelectrophoretic mobility of the analyte. If the electro-osmotic mobilityis written as μ_(EO)=μ_(EO) ⁰·η(20)/η(T), then by adjusting the ratio ofthe lengths of the hot and cold channels, (assuming ƒ_(EP)(T)=constant)focusing can be achieved if ƒ(cold)/ƒ(hot)<−μ₀/μ_(EO) ⁰<ƒ(hot)/ƒ(cold),where ƒ(hot)>ƒ(cold). If ƒ(hot)<ƒ(cold), then the inequalities have theopposite sign. If x is the fraction of the total channel length that ishot, then focusing will occur if: x·ƒ(hot)/ƒ(cold)+(1−x)<−μ₀/μ_(EO)⁰<x+(1−x)·ƒ(cold)/ƒ(hot), where ƒ(hot)>ƒ(cold). By adjusting x, it isthen possible to tune the range of analyte mobilities that are focused.

[0046] It should be noted that this can also be done for microchannelsof non-constant cross-section. The final results are essentiallyunchanged, since in most instances, the dependence on thecross-sectional area of the channel cancels out in the equations. As aresult, it is possible to generate the temperature gradient using Jouleheating within the microchannel. This would serve to simplify the designand operation of a microfluidic device using this technique evenfurther, since the focusing and the temperature gradient could beproduced using the same pair of electrodes as illustrated in FIG. 4(a).

[0047] Microchannel 20 shown schematically in FIG. 4(a), has electrodes,22, 24, and two sections, sections 26, 28, of different cross-sectionalarea. Section 26 has a cross-sectional area of 27 and section 28 has across-sectional area of 29. The electrical resistance per unit length ofeach section is given by: R₁=1/(σ·A₁), where σ is the conductivity ofthe buffer in the microchannel 20. When a current, I, is passed throughthe microchannel 20, the power per unit length dissipated through Jouleheating in each section will be: P₁=I²·R₁=I²/(σ·A₁). In general, theresulting temperature in section 28 will be higher than that in section26, as shown in FIG. 4(b): T₂>T₁. The electric field in each section ofthe microchannel 20 is given by the current multiplied by the resistanceper unit length:E_(i)=I·R_(i)=I/(σA_(i))=I·η(T_(i))·ƒ(T₁)/(σ₀·h(20)·A_(i)).

[0048] The electrophoretic velocity of an analyte in each section of thechannel is: u_(EP) ^(i)=μ₀·ƒ(T)₁)·I/(σ₀·ƒ_(EP)(T)·A₁). If a bulk flowvelocity is applied along the channel, it will not be the same in eachsection, but will instead be given by u_(B) ^(i)=u_(B) ⁰/A_(i), where,u_(B) ⁰ is a constant. The ratio of the electrophoretic velocity to thebulk velocity is then given by: u_(i)^(ratio)=μ₀·ƒ(T_(i))·I/(σ₀·ƒ_(EP)(T)·u_(B) ⁰)≡u₀^(ratio)·ƒ(T₁)/ƒ_(EP)(T) via adjusting u_(B) ⁰ so that |u₁^(ratio)|>1>|u₂ ^(ratio)| as shown in FIG. 4(d), which can result infocusing. Because the ratio of the electrophoretic velocity to the bulkvelocity does not depend on the cross-sectional areas of the twosections, the same considerations as above apply if bulk flow is appliedelectroosmotically.

[0049] One preferred buffer system is composed of 0.9 mol/L Trizma baseand 0.9 mol/L boric acid in water (1.8 M Tris/boric), with an expectedpH of about 8.7 (at room temperature). From measurements of theconductivity of the buffer, the function ƒ(T) was determined to varyfrom 1 at 20° C. to 0.77 at 70° C.

[0050] Joule heating may be used to generate the temperature gradient inthe microchannel device of FIG. 4(a). The following is a non-limitingexample demonstrating Joule heating of a microchannel of the type shownin FIG. 4 (a).

[0051] The microchannel used for this demonstration was similar to theone shown schematically in FIG. 4(a). The width, i.e., cross sectionalarea 29, of the narrow channel, i.e., section 28, was about 70 μm, andthe width of the wide section, i.e., section 26, was of the crosssectional area 26 was about 350 μm. The length of the tapered portion ofthe channel was about 500 μm. The depth of all portions of the channelwas about 30 μm. The total length of the microchannel was about 2 cm,with the length of the section 28 divided by the total length, x≅0.8.Access to each end of the microchannel was provided by a 3 mm holethrough the lid piece of the microchannel.

[0052] An 8 μmol/L solution of carboxyfluorescein in the 1.8 MTris/boric buffer was prepared. The analyte to be concentrated was thecarboxyfluorescein. Detection of the analyte was performed using afluorescence microscope and CCD cameras. Simultaneous color andgrayscale images were obtained.

[0053] To demonstrate gradient focusing using Joule heating, themicrochannel was filled with the caboxyfluorescein solution and 1900 Vwas applied along its length, with the positive voltage V₂ applied tothe narrow end via electrode 22, and the wide end held at ground atelectrode 24.

[0054] After 6 min., the carboxyfluorescein was highly concentrated atthe junction between sections 26 and 28 of the microchannel 20. Theconcentration factor achieved by using this example was typically about100-fold per minute.

[0055] Referring now to FIG. 5, in order to have better control of thetemperature gradient, experiments were done using three temperaturezones, two cold zones provided by cooling copper blocks 53 a, 53 bcovering much of the ends of the microchannel 50, and one hot zoneprovided by heated copper block 52. The microchannel 50 was made out ofthin (125 μm) sheets of poly(carbonate) substrate 51, which were pressedonto the copper blocks 52, 53 a, 53 b. Thermal contact between thepoly(carbonate) and the copper blocks was insured using a thermallyconductive adhesive 56. The copper blocks 52, 53 a, 53 b were arrangedso that there was a 1 mm gap 58 between the heated copper block 52 andthe cooling copper block 53 a and a 2 mm gap 59 between heated copperblock 52 and the cooling copper block 53 b.

[0056] Microchannel 50 also includes electrodes 55, 54, bufferreservoirs 57, and a narrow hot zone 50 a near the middle of themicrochannel 50. The heated copper block 52 was heated using a smallhigh-power resistor embedded into the copper and its temperature wasregulated using a PID temperature controller (Omega Engineering Inc,Stamford, Conn.). To regulate the temperature of the cold zones, ¼ inchdiameter holes were drilled through the cooling copper blocks 53 a, 53 band cold water from a thermostatted bath (Neslab, Portsmouth, N.H.) waspassed through them.

[0057] Thin polycarbonate microchannel chips, i.e. substrate 51 wasattached to the copper blocks 52, 53 a, 53 b using thermally conductiveadhesive 56 in the form of transfer tape (3M). The substrate 51 waspressed against the copper blocks 52, 53 a, 53 b from above with 3 mmthick PDMS (Sylgard 184, Dow Corning, Midland, Mich.) gaskets 60 and a 2mm thick acrylic (Acrylite OP-4, Cyro Industries, Mt. Arlington, N.J.))top plate 61, which was secured to the outer copper clocks using nylonscrews (not shown).

[0058] During temperature gradient focusing, a voltage potential isapplied to electrode 55 and electrode 54 is set to ground to allowmicrochannel 50 to provide focusing and separation of different types ofanalytes: small dye molecules, amino acids, proteins, DNA, colloidalparticles, and cells.

[0059] The microchannel 50 may be formed by imprinting with a micromachined silicon template and then sealed with a similar materialaccording to the method disclosed in Ross, D.; Gaitan, M.; Locascio, L.E., Analytical Chemistry 2001, 73, 4117-23, herein incorporated byreference.

[0060] The copper block arrangement was also used to determine thedegree of focusing that could ultimately be reached with temperaturegradient focusing. Beginning with a 8 nM solution of Oregon Green 488carboxylic acid in 1.8 M Tris/boric, 100 min of focusing resulted in afocused plug of Oregon Green 488 carboxylic acid with a peakconcentration over 80 μM—a greater than 10000-fold increase inconcentration.

[0061] It will become readily apparent to one of ordinary skill in theart that the present method provides for use in numerous applications.For example, temperature gradient focusing could be used as apreconcentration step before an analysis or separation or as asimultaneous concentration and separation technique.

[0062] In addition, temperature gradient focusing may be used with anycharged species in solution and not just small molecules. For example,the analytes may include larger molecules such as proteins and DNA, oreven particles and cells. Further, the present method can be used withparticles to create packed beds of particles or cells for use in otheranalysis steps. In addition, the present method can be adapted for useto sort particles or cells by electrophoretic mobility.

[0063] In one separation mode, the bulk velocity could be ramped overtime to scan focused sample peaks past a fixed detector, e.g. thedetector shown in FIG. 5. This would produce results similar tocapillary electrophoresis but the widths of the sample peaks would bedetermined by the applied gradients and the peak heights would bedetermined by how long a given peak was in the focusing “window”. If theramp speed were halved, the peak heights would all be doubled, so thatthe ramp rate could be chosen dependent on the concentration limit ofdetection necessary. Alternatively, the focusing window could remainfixed and a scanning or imaging detector could be used to locate theseparate peaks.

[0064] In a further embodiment, the method may be adapted for a systemwhere temperature dependence is due to something other than the ionicstrength. An example is a system having ƒ(T) constant but ƒ_(EP)(T) notconstant, or variable. One way to accomplish this would be to use abuffer with a temperature dependent pH. In such a system, thisembodiment of the present invention is similar to isoelectric focusingschemes. However, the present environment differs from isoelectricfocusing in that, in the present system, an opposing buffer flow isapplied so that analytes are focused at a pH other than theirisoelectric points.

[0065] When using any of the embodiments of the present method,operating parameters which include voltage, bulk flow rate, andtemperature of the different zones may be held constant with time orvaried with time to affect the position and width of focused samplepeaks. Varying of parameters may be accomplished using any of a numberof methods which include the methods previously described above in whichthe focused sample peaks are scanned past a fixed detector.

[0066] Advantageously, in order to achieve the fastest accumulation ofanalyte in the focused peak, the highest possible voltage should beused. However, a higher applied voltage requires a faster bulk flowwhich results in greater dispersion, i.e., wider focused peaks, which isdisadvantageous for separation and for achieving preconcentration of asample to a high concentration in a very narrow peak. Therefore, a highvoltage and fast bulk flow could be used for the initial accumulation ofanalyte into a relatively broad peak, and the voltage flow and flow ratecould be reduced to the point at which the peak is narrowest. Further,temperature gradients could be turned on and off to first concentratethe sample and then release the focused peak and allow it to flow ondown the channel. Further, the temperature gradient can be adjusted tobe linear or nonlinear, and the temperature gradient may be monotonic ornon-monotonic. Thus, operating parameters may be adjusted to achieve thedesired results.

[0067] While the previously disclosed embodiments are directed to amicrochannel or microfluid device, the present method may be adapted forincorporation for use with substantially larger channels which mayinclude millimeter and centimeter if not larger in dimension whichshould now be apparent to one of ordinary skill in the art. Becausetemperature gradient focusing uses low conductivity buffers, one canadapt the present method for use in much larger scale geometries thanthe micron-sized channels and capillaries described in detail herein.

[0068] Further, the previously described method can be adapted for usein modified capillary fluidic systems known to one of ordinary skill inthe art. FIG. 6 depicts a capillary fluidic system having a capillarytube 70 spanning between two buffer reservoirs 77. Two temperatureblocks, denoted as heated block 72 and cooling block 73 are locatedalong the length of the capillary tube 70 to provide a desiredtemperature gradient in the capillary tube 70. Alternatively,temperature blocks being both cooling, both heated, both at ambienttemperature, or any combination, thereof, may be substituted to providethe desired temperature gradient.

[0069] The buffer reservoirs 77 contain a buffer with temperaturedependent ionic strength. Electrodes 74, 75 are connected at one end toa power supply and on the other end, are in contact with the buffersolution in the buffer reservoirs 77. The power supply applies a drivingvoltage through the capillary tube 70. A source of bulk flow is driveneither by electro-osmosis with the applied driving voltage, by apressure gradient applied, e.g. by a pump, or a combination of the two.Detector 80 is used to detect analytes present in the buffer solution.

[0070] One of ordinary skill in the art now will readily appreciate thatthe present temperature gradient focusing differs from prior art methodssuch as sample stacking and isotachophoresis. In both cases, samples arefocused or concentrated as a result of gradients in theirelectrophoretic velocities. In sample stacking and isotachophoresis, thevelocity gradients are generated at the interfaces between buffers ofdifferent composition, and the point at which the concentration orfocusing occurs is not stationary, but moves along with theelectroosmotic flow in the channel or capillary. In contrast to bothsample stacking and isotachophoresis, the velocity gradients thatproduce analyte focusing in the present temperature gradient focusingresult from applied temperature gradients.

[0071] Further, one skilled in the art will recognized that the presenttemperature gradient focusing differs from isoelectric focusingtechniques such as those disclosed in U.S. Pat. Nos. 3,664,939 and5,759,370. Unlike isoelectric focusing techniques in which the pHgradient is established by using a buffer system that has a temperaturedependent pH, the present temperature gradient focusing utilizes abuffer that has a temperature dependent ionic strength. When atemperature gradient and a voltage are applied to a microchannel, theionic strength gradient of the buffer gives rise to a velocity gradient,which is used for focusing. As a result, an analyte present in thebuffer is focused at a point where the analyte's total velocity, i.e.,the sum of the electrophoretic velocity and the bulk velocity of thebuffer is zero. Therefore, in the present temperature gradient focusing,the pH and the isoelectric point of the analyte are not critical.

[0072] It will now be apparent to one of ordinary skill in the art thatthe present microfluidic device and temperature gradient focusing methodprovide numerous advantages over prior devices and methods. The presentdevice and method are simpler to implement as no imbedded electrodes orsalt bridges are necessary. In addition, like isoelectric focusing,temperature gradient focusing can be used to both concentrate andseparate analytes, but without the disadvantages associated withisoelectric focusing.

[0073] A further advantage of the present invention is provided in thatonly a single, continuous buffer system is required. Solid phaseextraction and related preconcentration methods of the prior art requiremultiple buffers where one buffer is used to carry the analyte to thepreconcentrator and a second buffer is used to release the analyte fromthe preconcentrator. Further examples of multiple buffer systems includesample stacking, field amplified injection, iosotachophoresis, andsweeping.

[0074] Further, the present temperature gradient focusing providesenhanced concentration when compared with the prior art of other singlepreconcentration methods.

[0075] Although the invention has been described above in relation topreferred embodiments thereof, it will be understood by those skilled inthe art that variations and modifications can be effected in thesepreferred embodiments without departing from the scope and spirit of theinvention.

What is claimed is:
 1. A method for directing ionic analytes contained in an ionic buffer solution of a system, said method comprising the steps of: producing an electric current flow in an ionic buffer solution containing at least one species of ionic analyte to cause the analyte ions to migrate electrophoretically; establishing, in said buffer solution, a temperature gradient having a significant component substantially aligned with said current flow thereby generating a gradient of the electrophoretic velocity of the analytes; and producing a bulk flow of said buffer solution to have a significant component substantially aligned in the direction opposite the direction of the electrophoretic migration of one or more of the analytes so that the total velocity of one or more of the analytes is equal to zero at some point in the system.
 2. The method of claim 1, wherein said temperature gradient establishes a gradient in the ionic strength of said buffer.
 3. The method of claim 1, wherein said temperature gradient establishes a gradient in the pH of said buffer, and whereby analytes are focused at a pH other than the isoelectric points of the respective analytes.
 4. The method of claim 1, wherein said temperature gradient establishes gradients in both the ionic strength and pH of the buffer, and whereby analytes are focused at a pH other than the isoelectric points of the respective analytes.
 5. The method of claim 1, wherein said temperature gradient is applied so as to produce an electrophoretic velocity gradient which concentrates analytes present in the ionic buffer.
 6. The method of claim 1, wherein said temperature gradient is applied so as to produce gradients in the electrophoretic velocities of the analytes present in the ionic buffer thereby causing different analytes to focus at different points within the buffer so as to separate the different analytes.
 7. The method of claim 1, wherein the analyte is selected from the group consisting of small ions, amino acids, DNA, particles, cells and proteins.
 8. The method of claim 1, wherein the bulk flow is generated by electroosmosis.
 9. The method of claim 1, wherein the bulk flow is generated by pressure gradients.
 10. The method of claim 1, wherein the bulk flow is generated by a combination of electroosmosis and pressure gradients.
 11. The method of claim 1, wherein at least one operational parameter selected from the group consisting of temperature, electric current, and bulk flow rate is varied over time to affect the position and width of focused sample peaks.
 12. The method of claim 1, wherein operational parameters consisting of temperature, electric current, and bulk flow rate are held constant.
 13. The method of claim 1, wherein the temperature gradient is one of linear and non-linear.
 14. The method of claim 1, wherein the temperature gradient is one of monotonic and non-monotonic.
 15. The method of claim 1, wherein the step of establishing a temperature gradient comprises applying an electric current to the buffer to produce the temperature gradient by Joule heating.
 16. The method of claim 1, wherein the ionic buffer is supplied as a continuous single buffer flow.
 17. The method of claim 1, wherein the buffer and analytes are contained within a microchannel.
 18. The method of claim 17, wherein the step of establishing a temperature gradient comprises supplying thermal energy to the microchannel via a heated block.
 19. The method of claim 17, wherein the step of applying a temperature gradient comprises cooling a portion of the microchannel using the ambient temperature as a maximum temperature.
 20. The method of claim 17, wherein the step of applying a temperature gradient comprises supplying thermal energy to the microchannel via a heated block and removing thermal energy from the microchannel via a cooled block.
 21. The method of claim 1, wherein the buffer and analytes are contained within a capillary tube.
 22. The method of claim 21, wherein the step of establishing a temperature gradient comprises supplying thermal energy to the capillary tube via a heated block.
 23. The method of claim 21, wherein establishing a temperature gradient comprises cooling a portion of the capillary tube using ambient temperature as a maximum temperature.
 24. The method of claim 21, wherein the step of establishing a temperature gradient comprises supplying thermal energy to the capillary tube via a heated block and removing thermal energy from the capillary tube via a cooled block.
 25. A fluidic device, comprising: a fluid conduit; an ionic buffer disposed in said fluid conduit; an electric current source for providing an electric current flow through said ionic buffer in said fluid conduit; at least one heat source or heat sink, thermally coupled to said fluid conduit, for providing for a temperature gradient having a significant component substantially aligned with said current flow so as to form an electrophoretic velocity gradient within said fluid conduit; and a source of bulk fluid flow for providing an opposing flow of said buffer in said fluid conduit.
 26. The fluid device of claim 25, wherein said ionic buffer has a temperature dependent ionic strength.
 27. The fluid device of claim 25, wherein said ionic buffer has a temperature dependent pH.
 28. The fluidic device of claim 25, wherein said fluid conduit comprises a microchannel formed in a substrate, having a geometry with at least one spatial dimension on the order of micrometers, and where a temperature gradient is applied to said substrate.
 29. The fluidic device of claim 25, wherein said at fluid conduit comprises a channel formed in a substrate and having a geometry with at least one spatial dimension on the order of at least one millimeter, and where a temperature gradient is applied to said substrate.
 30. The fluidic device of claim 25, wherein said fluid conduit comprises a channel formed in a substrate and having a geometry with at least one spatial dimension on the order of at least one centimeter, and where a temperature gradient is applied to said substrate.
 31. The fluidic device of claim 25, wherein said heat source comprises a power supply for applying an electrical current to said fluid conduit to thereby generate the temperature gradient in said buffer by Joule heating.
 32. The fluidic device of claim 25, wherein said at least one heat source comprises a heated block for providing thermal energy to said fluid conduit.
 33. The fluidic device of claim 25, wherein said at least one heat sink comprises a cooling block for removing thermal energy from said fluid conduit.
 34. The fluidic device of claim 32, wherein said at least one heat sink further comprises a cooling block spaced from said heated block and thermally coupled to said fluid conduit for removing thermal energy from said fluid conduit.
 35. The fluidic device of claim 32, further comprising a thermally conductive adhesive disposed between said heated block and said fluid conduit.
 36. The fluidic device of claim 33, wherein said heat source comprises a power supply for applying an electrical current to said fluid conduit to thereby generate the temperature gradient in said fluid conduit.
 37. The fluidic device of claim 25, wherein said fluid conduit comprises a capillary tube. 